2,3-butanediol, 2-butanone, and 2-butanol are important industrial chemicals. 2,3-butanediol may be used in the chemical synthesis of butene and butadiene, important industrial chemicals currently obtained from cracked petroleum, and esters of 2,3-butanediol may be used as plasticizers (Voloch et al. Fermentation Derived 2,3-Butanediol, in Comprehensive Biotechnology, Pergamon Press Ltd, England Vol 2, Section 3:933-947 (1986)). 2-Butanone, also referred to as methyl ethyl ketone (MEK), is a widely used solvent and is the most important commercially produced ketone, after acetone. It is used as a solvent for paints, resins, and adhesives, as well as a selective extractant, activator of oxidative reactions, and it can be chemically converted to 2-butanol by reacting with hydrogen in the presence of a catalyst (Nystrom, R. F. and Brown, W. G. (J. Am. Chem. Soc. (1947) 69:1198). Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase.
Microorganisms may be engineered for expression of biosynthetic pathways for production of 2,3-butanediol, 2-butanone, and/or 2-butanol. US Patent Pub US20070292927A1 discloses the engineering of recombinant microorganisms for expression of a biosynthetic pathway having 2,3-butanediol and 2-butanone as intermediates and 2-butanol as the end product. The pathway initiates with cellular pyruvate. Thus production of 2,3-butanediol, 2-butanone, and 2-butanol is limited by the availability of pyruvate substrate flow from natural host pathways into this engineered biosynthetic pathway.
In lactic acid bacteria, a limited amount of 2,3-butanediol may be produced naturally, but the major pyruvate metabolic pathway is conversion to lactate through activity of lactate dehydrogenase (LDH). Metabolic engineering to redirect pyruvate from lactate to other products in lactic acid bacteria has had unpredictable results. Production of alanine in LDH-deficient Lactococcus lactis expressing alanine dehydrogenase was shown by Hols et al. (Nature Biotech. 17:588-592 (1999). However, production of ethanol in LDH-deficient Lactobacillus plantarum expressing pyruvate decarboxylase was very limited, with carbon flow not significantly improved toward ethanol and lactate still produced (Liu et al. (2006) J. Ind. Micro. Biotech. 33:1-7).
Where a lactic acid bacteria is the preferred host for the production of 2-butanol and 2-butanone, a need exists therefore for lactic acid bacteria to have a tightly regulated carbon flow from pyruvate to 2,3-butanediol. To date no bacteria has been engineered to produce this advantage and the art suggests that simply reducing the carbon flow from pyruvate to lactate via lactate dehydrogenase may not be sufficient. Applicants have solved the stated problem through the unexpected discovery that introduction of a heterologous polypeptide having butanediol dehydrogenase activity in combination with reduction in endogenous lactate dehydrogenase results in unpredictably high rates of conversion of pyruvate to down stream products and particularly 2,3-butanediol.